The goal of this proposal is to develop improved oncolytic herpesviral vectors (oHSV) for the treatment of glioblastomas (GBM), a deadly cancer of the nervous system for which adequate therapy remains elusive Oncolytic viruses derive their anti-cancer activity from their ability to lyse cancer cells and spread within the tumor while sparing normal cells. Their selectivity for cancer cells is generally based on natural or engineered defects in the viral genome that inactivate key replication functions in normal cells but are complemented by the abnormal environment within cancer cells. HSV is an attractive vector for oncolytic therapy for tumors of the nervous system because of its natural neurotropism, high lytic activity, well-defined life cycle controlled by two essential genes, and capacity to deliver multiple exogenous anti-cancer protein products to enhance their efficacy. Clinical trials of current oncolytic HSV (oHSV) have demonstrated safety but limited efficacy, due in part to barriers examined by each of the projects of this program grant. Our study seeks to take advantage of new vector engineering technologies to achieve greater replication efficiency and oHSV spread within the tumor without compromising safety. We hypothesize that restricting oHSV infection to tumor cells by engineering steps referred to as retargeting, combined with measures to block virus replication in normal cells based on signature differences in microRNA expression profiles between normal and cancer cells, will provide a new generation of oHSVs that will prove both highly effective and safe. In Aim 1, we will redirect oHSV infectivity to glioblastoma cells by viral envelope engineering and in collaboration with Project 2-3, test, the effect of HDAC inhibition and virus-mediated ectopic chondroitinase expression on oHSV distribution and spread. In Aim 2, we will install genetic elements in the oHSV that will allow replication exclusively in glioblastoma cells as a function of the unique microRNA signatures of these cells. In Aim 3, animal experiments will be performed to test the safety and efficacy of selected oHSVs that combine key features from the first two aims. With potential further improvements based on feedback from all other projects of this program, we expect that we will engineer a promising new generation of oHSV for evaluation in clinical trials.